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PubMed1976

Comparative evaluation of drug interaction publications--1976.

The usefulness of several reference books in the retriveal and application of drug interaction information was evaluated. Each publication was evaluated as to whether it included information that would be necessary to determine if a suspected interaction was actually occurring. Twenty drug combinations which may interact were studied. In addition, the general format and features of each publication were reviewed. The American Pharmaceutical Association's Evaluations of Drug Interactions and Hansten's Drug Interactions were reviewed favorably on all points. Stockley's Drug Interactions and their Mechanisms was considered nearly as complete and certainly as authoritative. Cohen's Drug Interactions: A Handbook for Clinical Use, Gant's Drug Interaction Index: A Survey of Drug Interactions and Hartshorn's Handbook of Drug Interactions, were deemed useful as an added check if an interaction suspected clinically was not included in any of the former group.

Drug Information Services↗
PubMed1998

Oxazole yellow dye interactions with short DNA oligomers of homogeneous base composition and their hybrids.

Interactions between short single-stranded DNA oligomers of homogeneous base composition and the fluorescent probes oxazole yellow (YO) and its homodimer YOYO are described. The oligomers included 15-mers and 30-mers of polydA, polydT, polydG, and polydC. Interactions between the dyes and DNA hybrids formed from complementary homogeneous strands of equal length were also investigated. No interactions were observed between the dyes and the monomeric monophosphate nucleosides A, G, T, or C. The dyes were found to interact much more strongly with the purine oligomers polydA and polydG than with the pyrimidine oligomers polydT and polydC. PolydA of both lengths has strong interactions with YOYO, whereas the polydG 30-mer interacts strongly with monomeric YO. The 15-mers of polydG and polydC of both lengths show little interaction with either dye. Interactions of the dyes with the polydA/polydT and polydG/polydC hybrids tend to be dominated by interactions with polydA and polydG, respectively. Although dye interactions generally were facilitated by hybridization, particularly for polydA/polydT, the interactions were similar to those with the single strands and different from those that have been observed in long double-stranded DNA.

Base Composition↗
PubMed2004

The nature of intermolecular CuI...CuI interactions: a combined theoretical and structural database analysis.

The nature of intermolecular interactions between dicoordinate Cu(I) ions is analyzed by means of combined theoretical and structural database studies. Energetically stable Cu(I).Cu(I) interactions are only found when the two monomers involved in the interaction are neutral or carry opposite charges, thus allowing us to speak of bonding between the components of the bimolecular aggregate. A perturbative evaluation of the components of the intermolecular interaction energies, by means the IMPT scheme of Stone, indicates that both the Coulombic and dispersion forces are important in determining the Cu(I).Cu(I) bonding interactions, because only a small part of that energy is attributable to Cu.Cu interactions, while a large component results from Cu.ligand interactions. The electrostatic component is the dominant one by far in the interaction between charged monomers, while in the interaction between neutral complexes, the electrostatic component is found to be of the same order of magnitude as the dispersion term. Bimolecular aggregates that have like charges are repulsive by themselves, and their presence in the solid state results from anion.cation interactions with ions external to this aggregate. In these cases, the short-contact Cu.Cu interactions here should be more properly called counterion-mediated Cu.Cu bonds.

Journal Article↗
PubMed2006

Isostructurality, polymorphism and mechanical properties of some hexahalogenated benzenes: the nature of halogen...halogen interactions.

The nature of intermolecular interactions between halogen atoms, X...X (X = Cl, Br, I), continues to be of topical interest because these interactions may be used as design elements in crystal engineering. Hexahalogenated benzenes (C6Cl(6-n)Br(n), C6Cl(6-n)I(n), C6Br(6-n)I(n)) crystallise in two main packing modes, which take the monoclinic space group P2(1)/n and the triclinic space group P1. The former, which is isostructural to C6Cl6, is more common. For molecules that lack inversion symmetry, adoption of this monoclinic structure would necessarily lead to crystallographic disorder. In C6Cl6, the planar molecules form Cl...Cl contacts and also pi...pi stacking interactions. When crystals of C6Cl6 are compressed mechanically along their needle length, that is, [010], a bending deformation takes place, because of the stronger interactions in the stacking direction. Further compression propagates consecutively in a snakelike motion through the crystal, similar to what has been suggested for the motion of dislocations. The bending of C6Cl6 crystals is related to the weakness of the Cl...Cl interactions compared with the stronger pi...pi stacking interactions. The triclinic packing is less common and is restricted to molecules that have a symmetrical (1,3,5- and 2,4,6-) halogen substitution pattern. This packing type is characterised by specific, polarisation-induced X...X interactions that result in threefold-symmetrical X3 synthons, especially when X = I; this leads to a layered pseudohexagonal structure in which successive planar layers are inversion related and stacked so that bumps in one layer fit into the hollows of the next in a space-filling manner. The triclinic crystals shear on application of a mechanical stress only along the plane of deformation. This shearing arises from the sliding of layers against one another. Nonspecificity of the weak interlayer interactions here is demonstrated by the structure of twinned crystals of these compounds. One of the compounds studied (1,3,5-tribromo-2,4,6-triiodobenzene) is dimorphic, adopting both the monoclinic and triclinic structures, and the reasons for polymorphism are suggested. To summarise, both chemical and geometrical models need to be considered for X...X interactions in hexahalogenated benzenes. The X...X interactions in the monoclinic group are nonspecific, whereas in the triclinic group some X...X interactions are anisotropic, chemically specific and crystal-structure directing.

Journal Article↗
PubMed1995

Interactions between the amino-terminal domain of p56lck and cytoplasmic domains of CD4 and CD8 alpha in yeast.

The interactions between CD4 or CD8 and p56lck were tested using the two-hybrid protein interaction system in yeast. Plasmid constructs were created which fuse the cytoplasmic domains of either CD4 or CD8 alpha to the DNA-binding protein LexA, and the unique amino-terminal domain of p56lck fused to a transcriptional activation domain. These constructs were transfected into yeast bearing lacZ and LEU2 reporter genes controlled by upstream LexA operator sequences. Yeast transfectants bearing either CD4 or CD8 alpha hybrid proteins in combination with the amino terminal p56lck hybrid protein exhibited increased beta-galactosidase activity and growth on leucine-deficient medium, indicating interactions between these protein domains. Quantitation of reporter activation indicated that the interaction of p56lck with CD8 alpha is at least 18-fold weaker than the interaction with CD4 in this assay. This reduced interactive capacity is apparently not due to competition by CD8 alpha interacting with itself, since homotypic or heterotypic interactions between CD8 alpha and/or CD4 could not be detected. Truncation and point mutants demonstrated that the interactions of p56lck with CD4 or CD8 alpha were dependent on the integrity of a pair of cysteines on each protein. The results indicate that these interactions do not require any additional proteins. Additionally, expression of the entire p56lck molecule as a hybrid with LexA resulted in dramatic reduction in the growth of yeast. Though the two-hybrid system is a powerful tool for examining protein interactions, this result indicates potential limitations in studying full-length src family tyrosine kinases in yeast.

Amino Acid Sequence↗
PubMed1998

Interactions among HIV gp120, CD4, and CXCR4: dependence on CD4 expression level, gp120 viral origin, conservation of the gp120 COOH- and NH2-termini and V1/V2 and V3 loops, and sensitivity to neutralizing antibodies.

The binding of HIV-derived recombinant soluble (s)gp120 to the CD4(+)/CXCR4(+) A3.01 T cell line inhibits the binding of the CXCR4-specific monoclonal antibodies 12G5, which interacts with the second extracellular loop, and 6H8, which binds the NH2 terminus. We have used this as an assay to analyse the interaction of recombinant sgp120 from diverse viral origins with CXCR4. The strength of the interaction between sgp120 and CXCR4 correlated with sgp120 affinity for the CD4-CXCR4 complex, and the interaction of sgp120MN and sgp120IIIB with CXCR4 was highly dependent on the level of CD4 expressed on a variety of different T cell lines. sgp120 from X4, R5X4, and R5 viruses interacted with CXCR4, although the R5 sgp120-CXCR4 interactions were weaker than those of the other gp120s. The interaction of sgp120IIIB or sgp120MN with CXCR4 was inhibited by neutralizing monoclonal antibodies that prevent the sgp120-CD4 interaction but also by antibodies specific for the gp120 V2 and V3 loops, the CD4-induced epitope and the 2G12 epitope, which interfere weakly or not at all with CD4-sgp120 binding. The binding to A3.01 cells of wild-type sgp120HxB2, but not of sgp120 deleted in the COOH and NH2 termini, interfered with 12G5 binding in a dose-dependent manner. Further deletion of the V1 and V2 loops restored CXCR4 binding activity, but additional removal of the V3 loop eliminated the gp120-CXCR4 interaction, without decreasing the affinity between mutated sgp120 and CD4. Taken together, these results demonstrate that the interactions between sgp120 and CXCR4 are globally similar to those previously observed between sgp120 and CCR5, with some apparent differences in the strength of the sgp120-CXCR4 interactions and their dependence on CD4.

Antibodies, Monoclonal↗
PubMed1994

Sensory modulation of synchronous thalamocortical interactions in the somatosensory system of the cat.

Neuronal responses to hairy skin stimulation were simultaneously recorded in the ventral posterolateral nucleus (VPL) of the thalamus and primary somatosensory cortex (SI) of halothane-anesthetized cats. Among 233 thalamocortical neuron pairs, cross-correlation analysis revealed significant interactions in 120 pairs. Excitatory interactions were most prevalent and included influences occurring exclusively in the thalamocortical (41 pairs) or corticothalamic (23 pairs) directions as well as multiphasic interactions (40 pairs) in both directions. Only 16 pairs exhibited inhibitory interactions and 7 of these involved multiphasic combinations of excitation and inhibition. In 14 of these neuron pairs, inhibition was exerted in the corticothalamic direction. Receptive field (RF) overlap between thalamic and cortical neurons varied considerably, and neuronal interactions were more likely for neuron pairs sharing large portions of their combined RFs. Computer-controlled stimulation was delivered to multiple RF sites but only 46% of the neuron pairs displayed interactions at more than one stimulation site and only four neuron pairs showed interactions at all stimulus positions. When interactions occurred at multiple stimulus sites, 40% of these interactions were characterized by timing shifts where the time interval between VPL and SI discharges varied as much as 20 ms because of stimulus relocation. In nine neuron pairs, systematic shifts in stimulus position produced reversals in the temporal sequence of thalamic and cortical neuronal discharges. Functional interactions between thalamic and cortical neurons were detected during both spontaneous and stimulus-induced activity. Matched-sample comparisons of connection strength and half-widths of thalamocortical peaks during spontaneous and stimulus-induced activity indicated that functional interactions produced by cutaneous stimulation were significantly stronger and had less temporal variability than those occurring spontaneously.

Animals↗
PubMed2004

Structural analysis of cation-pi interactions in DNA binding proteins.

Cation-pi interactions play an important role in the stability of protein structures. In this work, we have analyzed the influence of cation-pi interactions in DNA binding proteins. We observed cation-pi interactions in 45 out of 62 DNA binding proteins and there is no significant correlation between the number of amino acid residues and number of cation-pi interactions. These interactions are mainly formed by long-range contacts, and the role of short and medium-range contacts is minimal. The preference of Arg is higher than Lys to form cation-pi interactions. The pair-wise cation-pi interaction energy between aromatic and positively charged residues shows that Arg-Tyr energy is the strongest among the possible six pairs. The structural analysis of cation-pi interaction forming residues shows that Lys, Trp, and Tyr prefer to be in the binding site of protein-DNA complexes. Further, the accessible surface areas of cation-pi interaction forming cationic residues are significantly less than that of other residues. The preference of cation-pi interaction forming residues in different secondary structures shows that Lys prefers to be in strand and Phe prefers to be in turn regions. The results obtained in the present study will be useful in understanding the contribution of cation-pi interactions to the stability and specificity of protein-DNA complexes.

Amino Acids↗
PubMed2003

The relationship between sequence and interaction divergence in proteins.

There is currently a gap in knowledge between complexes of known three-dimensional structure and those known from other experimental methods such as affinity purifications or the two-hybrid system. This gap can sometimes be bridged by methods that extrapolate interaction information from one complex structure to homologues of the interacting proteins. To do this, it is important to know if and when proteins of the same type (e.g. family, superfamily or fold) interact in the same way. Here, we study interactions of known structure to address this question. We found all instances within the structural classification of proteins database of the same domain pairs interacting in different complexes, and then compared them with a simple measure (interaction RMSD). When plotted against sequence similarity we find that close homologues (30-40% or higher sequence identity) almost invariably interact the same way. Conversely, similarity only in fold (i.e. without additional evidence for a common ancestor) is only rarely associated with a similarity in interaction. The results suggest that there is a twilight zone of sequence similarity where it is not possible to say whether or not domains will interact similarly. We also discuss the rare instances of fold similarities interacting the same way, and those where obviously homologous proteins interact differently.

Amino Acid Sequence↗
PubMed2005

Mutational analysis demonstrates that specific electrostatic interactions can play a key role in the denatured state ensemble of proteins.

The nature of the denatured state ensemble has been controversial for decades owing, in large part, to the difficulty in characterizing the structure and energetics of denatured state interactions. There is increasing evidence for relatively non-specific hydrophobic clustering in the denatured states of some proteins but other types of interactions are much less well characterized. Here, we report the characterization of highly specific electrostatic interactions in the denatured state of a small alpha-beta protein, the N-terminal domain of the ribosomal protein L9 (NTL9). Mutation of Lys12 to Met has been shown to increase the stability of NTL9 significantly through the disruption of denatured state interactions. Here, we describe the analysis of the pH-dependent stability of 13 mutants designed to probe the nature of the Lys12 denatured state interaction. Lys12 is located in a lysine-rich region of the protein but analysis of a set of Lys to Met mutants shows that it plays a unique role in the denatured state. Analysis of mutants of all of the acidic residues in NTL9 shows that Lys12 forms a specific non-native electrostatic interaction with Asp8 in the denatured state ensemble. Thus the distribution of charge-charge interactions in the denatured state ensemble of NTL9 appears to be biased by few key interactions and is very different from that expected in a random coil. We propose that these interactions are not encoded by local sequence effects but rather reflect interactions among residues more distant in sequence. These results demonstrate that electrostatic as well as hydrophobic interactions can play an important role in the denatured state ensemble.

Hydrogen-Ion Concentration↗
PubMed2005

Probing bacterial interactions: integrated approaches combining atomic force microscopy, electron microscopy and biophysical techniques.

Recent developments in the application of Atomic Force Microscopy (AFM) and other biophysical techniques for the study of bacterial interactions and adhesion are discussed in the light of established biological and microscopic approaches. Whereas molecular-biological techniques combined with electron microscopy allow the identification and localization of surface constituents mediating bacterial interactions, with AFM it has become possible to actually measure the forces involved in bacterial interactions. Combined with the flexibility of AFM in probing various types of physical interactions, such as electrostatic interactions, specific ligand-receptor interactions and the elastic forces of deformation and extension of bacterial surface polymers and cell wall, this provides prospects for the elucidation of the biophysical mechanism of bacterial interaction. However, because of the biochemical and a biophysical complexity of the bacterial cell wall, integrated approaches combining AFM with electron microscopy and biophysical techniques are needed to elucidate the mechanism by which a bacterium interacts with a host or material surface. The literature on electron microscopy of the bacterial cell wall is reviewed, with particular emphasis on the staining of specific classes of cell-wall constituents. The application of AFM in the analysis of bacterial surfaces is discussed, including AFM operating modes, sample preparation methods and results obtained on various strains. For various bacterial strains, the integration of EM and AFM data is discussed. Various biophysical aspects of the analysis of bacterial surface structure and interactions are discussed, including the theory of colloidal interactions and Bell's theory of cell-to-cell adhesion. An overview is given of biophysical techniques used in the analysis of the properties of bacterial surfaces and bacterial surface constituents and their integration with AFM. Finally, we discuss recent progress in the understanding of the role of bacterial interactions in medicine within the framework of the techniques and concepts discussed in the paper.

Bacteria↗
PubMedDate not supplied

Interactive behavior in bipolar manic and schizophrenic patients and its link to thought disorder.

The research was designed to assess whether bipolar manic patients are unique in showing excessive interactive behavior and whether interactive behavior is linked to positive thought disorder in mania and other psychotic disorders. We compared the interactive behavior of manic patients versus schizophrenics and nonpsychotic patients. Degree of interactive behavior and severity of thought disorder were assessed in an inpatient sample of 140 patients, including 38 bipolar manic patients, and control samples of 40 acute schizophrenic patients, 32 nonpsychotic patients, and 30 chronically hospitalized schizophrenic patients. The assessment battery used standardized tests to assess interactive behavior and thought disorder. Manic patients displayed significantly more interactive behavior on all three tests than the other three patient groups. Interactive behavior made a greater contribution to the thought disorder of manic patients than to the thought disorder of the other three diagnostic groups. The other two acute patient groups also showed relationships between interactive behavior and thought disorder. The chronic schizophrenics did not show this pattern. The data indicated that many types of acute patients show some interactive behavior, but bipolar manic patients demonstrate more excessive interactive behavior than do other patients. There is a closer link between excessive interactive behavior and thought disorder in manic patients.

Acute Disease↗
PubMed2000

Identification of an interaction between the angiotensin II receptor sub-type AT2 and the ErbB3 receptor, a member of the epidermal growth factor receptor family.

To identify the proteins that interact and mediate angiotensin II receptor AT2-specific signaling, a random peptide library was screened by yeast-based Two-Hybrid protein-protein interaction assay technique. A peptide that shared significant homology with the amino acids located between the residues Gly-Xaa-Gly-Xaa-Xaa-Gly721 and Lys742, the residues predicted to be important for ATP binding of the ErbB3 and ErbB2 receptors, was identified to be interacting with the AT2 receptor. The interaction between the human ErbB3 receptor and the AT2 receptor was further confirmed using the cytoplasmic domain (amino acids 671-782) of the human ErbB3 receptor. Moreover, an AT2 receptor peptide that spans the amino acids 226-363, (spans the third ICL and carboxy terminal domain) could also interact with the AT2 receptor in a yeast Two-Hybrid protein-protein interaction assay. Studies using mutated and chimeric AT2 receptors showed that replacing the third intracellular loop (ICL) of the AT2 receptor with that of the AT1 abolishes the interaction between the ErbB3 and the AT2 in yeast Two-Hybrid protein-protein interaction assay. Thus the interaction between the AT2 receptor and the ErbB3 receptor seems to require the region spanning the third ICL and carboxy terminus of the AT2 receptor. Since the third ICL of the AT2 receptor is essential for exerting its inhibitory effects on cell growth, possible involvement of this region in the interaction with the cytoplasmic domain of the ErbB3 receptor suggests a novel signaling mechanism for the AT2 receptor mediated inhibition of cell growth. Furthermore, since both the AT2 and the ErbB3 receptors are expressed during fetal development, we propose that the existence of direct interaction between these two receptors may play a role in the regulation of growth during the initial stages of development.

Amino Acid Sequence↗
PubMed2003

Hepatitis C virus--cell interactions and their role in pathogenesis.

In summary, HCV-cell interactions include those directly involved with the HCV life cycle such as virus attachment, entry, and replication. Included within this broad area of research are the interactions of HCV proteins with the IFN system, cytokine and chemokine pathways such as IL-8, and various other cellular proteins and pathways. The plethora of contradictory and sometimes confusing accessory HCV-host interactions defies precise predictions of their role in HCV biology. It is clear that these virus-cell interactions affect HCV replication, antiviral resistance, persistence, and pathogenesis. Because HCV-host interactions are initiated immediately on infection, they are operative during acute HCV infection, whereby HCV interacts with innate cellular antiviral and immune systems. The magnitude and duration of these HCV-host interactions therefore may influence the development of acquired immunity. Because HCV exists as a quasispecies in all infected individuals, heterogeneity in biological responses to HCV-host interactions is predicted, revealing opportunities for the development of various genotypic and phenotypic prognostic indicators. With the model systems in place, these hypotheses can be tested. The challenge for the future is to determine if there is a hierarchical importance to these interactions, to delineate how these virus-cell interactions affect the patient infected with HCV, and to determine whether any of these interactions represents a target for therapeutic intervention.

Animals↗
PubMed2001

Protein splicing-based reconstitution of split green fluorescent protein for monitoring protein-protein interactions in bacteria: improved sensitivity and reduced screening time.

In this research, an improved detection system is described that allows an easy in vivo screening and selection of functional interactions between two interacting proteins in bacteria. We earlier reported a new concept for detecting protein-protein interactions based on reconstitution of split-enhanced green fluorescent protein (EGFP) by protein splicing (Ozawa, T.; et al. Anal. Chem. 2000, 72, 5151-5157.): Two putative interacting proteins are genetically fused to the split VDE inteins, which are linked directly to the N- and C-terminal halves of the split EGFP. Association of the interacting proteins results in functional complementation of VDE and protein-splicing reaction that leads to formation of an EGFP fluorophore. This technique simplified detection of protein interactions, but because of the low splicing efficiency of VDE intein, its sensitivity and screening time were not enough for detecting the protein interactions directly in living cells. In this paper, we have explored the use of the DnaE split intein from Synechocystis sp. PCC6803 for intracellular reconstitution of the split EGFP. We examined efficiency of the fluorophore formation by preparing four different split-EGFP types, among which EGFP dissected at the position between 157 and 158 was found to show the strongest fluorescence intensity upon protein interactions. A time required for the formation of EGFP after protein interactions was only 4 h, as compared to 3 days with the VDE intein. The protein interactions were thereby detected by an in vivo selection and screening assay in Escherichia coli on Luria broth agar plates. This improvement permits versatile designs of screening procedures either for ligands that bind to particular proteins or for molecules or mutations that block particular interactions between two proteins of interest.

Bacterial Proteins↗
PubMed1991

Analysis of the interaction between charged side chains and the alpha-helix dipole using designed thermostable mutants of phage T4 lysozyme.

It was shown previously that the introduction of a negatively charged amino acid at the N-terminus of an alpha-helix could increase the thermostability of phage T4 lysozyme via an electrostatic interaction with the "helix dipole" [Nicholson, H., Becktel, W. J., & Matthews, B. W. (1988) Nature 336, 651-656]. The prior report focused on the two stabilizing substitutions Ser 38----Asp (S38D) and Asn 144----Asp (N144D). Two additional examples of stabilizing mutants, T109D and N116D, are presented here. Both show the pH-dependent increase in thermal stability expected for the interaction of an aspartic acid with an alpha-helix dipole. Control mutants were also constructed to further characterize the nature of the interaction with the alpha-helix dipole. High-resolution crystal structure analysis was used to determine the nature of the interaction of the substituted amino acids with the end of the alpha-helix in both the primary and the control mutants. Control mutant S38N has stability essentially the same as that of wild-type lysozyme but hydrogen bonding similar to that of the stabilizing mutant S38D. This confirms that it is the electrostatic interaction between Asp 38 and the helix dipole, rather than a change in hydrogen-bonding geometry, that gives enhanced stability. Structural and thermodynamic analysis of mutant T109N provide a similar control for the stabilizing replacement T109D. In the case of mutant N116D, there was concern that the enhanced stability might be due to a favorable salt-bridge interaction between the introduced aspartate and Arg 119, rather than an interaction with the alpha-helix dipole. The additivity of the stabilities of N116D and R119M seen in the double mutant N116D/R119M indicates that favorable interactions are largely independent of residue 119. As a further control, Asp 92, a presumed helix-stabilizing residue in wild-type lysozyme, was replaced with Asn. This decreased the stability of the protein in the manner expected for the loss of a favorable helix dipole interaction. In total, five mutations have been identified that increase the thermostability of T4 lysozyme and appear to do so by favorable interactions with alpha-helix dipoles. As measured by the pH dependence of stability, the strength of the electrostatic interaction between the charged groups studied here and the helix dipole ranges from 0.6 to 1.3 kcal/mol in 150 mM KCl. In the case of mutants S38D and N144H, NMR titration was used to measure the pKa's of Asp 38 and His 144 in the folded structures.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed2004

Transferrin's mechanism of interaction with receptor 1.

The kinetics and thermodynamics of the interactions of transferrin receptor 1 with holotransferrin and apotransferrin in neutral and mildly acidic media are investigated at 37 degrees C in the presence of CHAPS micelles. Receptor 1 interacts with CHAPS in a very fast kinetic step (<1 micros). This is followed in neutral media by the interaction with holotransferrin which occurs in two steps after receptor deprotonation, with a proton dissociation constant (K(1a)) of 10.0 +/- 1.5 nM. The first step is detected by the T-jump technique and is associated with a molecular interaction between the receptor and holotransferrin. It occurs with a first-order rate constant (k(-1)) of (1.6 +/- 0.2) x 10(4) s(-1), a second-order rate constant (k(1)) of (3.20 +/- 0.2) x 10(10) M(-1) s(-1), and a dissociation constant (K(1)) of 0.50 +/- 0.07 microM. This step is followed by a slow change in the conformation with a relaxation time (tau(2)) of 3400 +/- 400 s and an equilibrium constant (K(2)) of (4.6 +/- 1.0) x 10(-3) with an overall affinity of the receptor for holotransferrin [(K'1)(-1)] of (4.35 +/- 0.60) x 10(8) M(-1). Apotransferrin does not interact with receptor 1 in neutral media, between pH 4.9 and 6, it interacts with the receptor in two steps after a receptor deprotonation (K(2a) = 2.30 +/- 0.3 microM). The first step occurs in the range of 1000-3000 s. It is ascribed to a slow change in the conformation which rate-controls a fast interaction between apotransferrin and receptor 1 with an overall affinity constant [(K(3))(-1)] of (2.80 +/- 0.30) x 10(7) M(-1). These results imply that receptor 1 probably exists in at least two forms, the neutral species which interacts with holotransferrin and not with apotransferrin and the acidic species which interacts with apotransferrin. At first, the interaction of the neutral receptor with holotransferrin is extremely fast. It is followed by the slow change in conformation, which leads to an important stabilization of the thermodynamic structure. In the acidic media of the endosome, the interaction of apotransferrin with the acidic receptor is sufficiently strong and rate-controlled by a very slow change in conformation which allows recycling back to the plasma membrane.

Antigens, CD↗
PubMed2003

Folding of a stable DNA motif involves a highly cooperative network of interactions.

Hairpins are structural elements that play important roles in the folding and function of RNA and DNA. The extent of cooperativity in folding is an important aspect of the RNA folding problem. We reasoned that an investigation into the origin of cooperativity might be best carried out on a stable nucleic acid system with a limited number of interactions, such as a stable DNA hairpin loop. The stable d(cGNAg) hairpin loop motif (closing base pair in lower case; loop in upper case; N = A, C, G, or T) is stabilized through only three interactions: two loop-loop hydrogen bonds in a sheared GA base pair and a loop-closing base pair interaction. Herein, we investigate this network of interactions and test whether the loop-loop and loop-closing base pair interactions communicate. Thermodynamic measurements of nucleotide analogue substituted oligonucleotides were used to probe the additivity of the interactions. On the basis of double mutant cycles, all interactions were found to be nonadditive and interdependent, suggesting that loop-loop and loop-closing base pair interactions form in a highly cooperative manner. When double mutant cycles were repeated in the absence of the other interaction, nonadditivity was significantly reduced suggesting that coupling is indirect and requires all three interactions in order to be optimal. A cooperative network of interactions helps explain the structural and energetic bases of stability in certain DNA hairpins and paves the way for similar studies in more complex nucleic acid systems.

DNA↗